35 effect of 6-dimethylaminopurine treatment duration on pronuclear formation and in vivo development of canine cloned embryo.

Artificial activation is an important step for successful somatic cell nuclear transfer (SCNT). In order to clone animals, diverse methods of activation have been studied to increase the developmental efficiency of cloned embryos. Here, we investigated the pronucleus formation and in vivo development of canine cloned embryos produced by different durations of 1.9mM 6-dimethylaminopurine (DMAP) treatment. For canine SCNT, in vivo-matured oocytes were enucleated, microinjected into the perivitelline space with donor cells, and fused by electrical stimulation. For activation, the fused couplets were cultured for 4min in 10μM calcium ionophore, and then they were divided into 2 groups: (1) the 2DMAP group was cultured for 2h in DMAP; (2) the 4DMAP group was cultured for 4h in DMAP. Activated cloned embryos were subjected to 2 analyses: (1) observing the pronuclear formation by bromodeoxyuridine (BrdU) incorporation at 2h, 4h and 8h post-activation (hpa), and (2) following fetus formation and pregnancy efficiency after embryo transfer into naturally synchronous recipients. Pregnancy diagnosis was performed by ultrasonography on Day 26 of embryo transfer. Data were analysed using Graph Prism software (GraphPad Software Inc., San Diego, CA, USA). All cloned embryos of the 2DMAP group showed BrdU incorporation at 2 hpa, whereas 4DMAP embryos showed 77.7% BrdU incorporation at 2 hpa (P<0.05). Incorporation of BrdU was detected in all cloned embryos of both experimental groups after 4 hpa and 8 hpa. A total of 370 cloned embryos were transferred to 24 surrogate mothers (182 cloned embryos into 12 recipients in 2DMAP group and 188 cloned embryos into 12 recipients in 4DMAP group). There was no significant difference in pregnancy rate (2DMAP; 41.6% v. 4DMAP; 33.3%) or implantation rates (2DMAP; 4.9% v. 4DMAP; 3.7%) between the 2 groups. In conclusion, DMAP exposure for 2h in activation completed pronucleus development of canine reconstructed embryos. However, none of the applied tested treatments resulted in increased implantation rates.